HUP0103484A2 - Combination therapies for b-cell lymphomas comprising administration of anti-cd20 antibody - Google Patents

Abstract

The subject of the invention is the use of anti-CD20 antibodies or their fragments in the treatment of B-cell lymphomas, more specifically the use of such antibodies or antibody fragments in combination medical procedures. The solution according to the invention is suitable for the treatment of B-cell lymphomas - mainly non-Hodgkins lymphoma. HE

Description

92196-5397/FRI

PUBLICATION COPY

Use of anti-CD20 antibody for combination therapy of B-cell lymphomas

The invention relates to the use of anti-CD20 antibodies or fragments thereof in the treatment of B-cell lymphomas/more particularly to the use of such antibodies or antibody fragments in combination therapy methods.

The solution according to the invention is suitable for the treatment of B-cell lymphomas - primarily non-Hodgkin's lymphoma.

The use of antibodies against the CD20 antigen as diagnostic and/or therapeutic agents for B-cell lymphoma has been previously described. CD20 is a useful marker or target molecule for B-cell lymphomas because it is expressed at very high levels on the surface of malignant B-cells (B-cells whose continued proliferation can lead to the development of B-cell lymphoma).

CD20 or Bp35 is a B-lymphocyte-restricted differentiation antigen that is expressed early in B-cell development and persists until differentiation into plasma cells. Some have suggested that the CD20 molecule may regulate a step in the B-cell activation process required for cell cycle initiation and differentiation. However, as noted, CD20 is usually expressed at very high levels on neoplastic B cells.

Our file number: 93989-6838c/PÁ

- 2 on the surface of cells. The CD20 antigen seems suitable for targeted therapy because it is not rejected, does not change, and does not enter the cell interior.

Previous descriptions of therapeutic methods using CD20 antibodies have included administration of the therapeutic anti-CD20 antibody alone or in combination with a second, radiolabeled anti-CD20 antibody or therapeutic agent.

The FDA (Food and Drug Administration) has approved the use of one such anti-CD20 antibody, Rituxan®, for the treatment of relapsed and previously treated low-grade non-Hodgkin lymphoma (NHL). In addition, Rituxan® in combination with radiolabeled murine anti-CD20 antibody has been recommended for the treatment of B-cell lymphoma.

However, while anti-CD20 antibodies, and in particular Rituxan® (US brand name, MabThera® in the UK, Rituximab® elsewhere), have been reported to be successful in the treatment of B-cell lymphomas, such as non-Hodgkin lymphoma, many patients have experienced relapse of the disease. Therefore, more effective treatments are needed.

In particular, it would be advantageous if anti-CD20 antibodies were used in combination with other lymphoma treatments and if there were a possibility of developing combination therapies that reduce the chance or frequency of relapse. It would be advantageous to further develop current lymphoma treatment protocols.

- 3 development, which would allow patients who do not respond to other treatments to be treated with chimeric or radiolabeled anti-CD20 antibodies. It would also be useful if therapeutic intervention with anti-CD20 antibodies, and especially their combination with other treatments, could be used to treat other types of lymphomas in addition to low-grade follicular non-Hodgkin lymphoma.

The invention relates to combination therapies for B-cell lymphomas and to the benefits of treating relapsed or refractory B-cell lymphomas with chimeric and radiolabeled anti-CD20 antibodies. We have shown that treatment with anti-CD20 antibodies, when used in combination with cytokines, radiotherapy, myeloablative therapy or chemotherapy, has a beneficial synergistic effect. Surprisingly, patients who have previously undergone bone marrow or stem cell transplantation have shown an increased response to treatment compared to patients who have not been treated before.

The solution according to the invention is described in detail below.

The invention relates to combination methods for the treatment of B-cell lymphomas. In general, these are methods for the treatment of relapsed B-cell lymphoma, in which a patient who has previously received anti-lymphoma treatment but has relapsed is treated with a therapeutically effective dose of a chimeric anti-CD20 antibody. The prior anti-lymphoma treatment may be, for example, anti- 4 • * · · · • · · · * · · ·

Treatment with CD20 antibodies, bone marrow or stem cell transplantation, radiotherapy or chemotherapy. Prior chemotherapy may refer to treatment with various chemotherapeutic agents and combination treatment protocols, such as CHOP, ICE, mitozantrone, cytarabine, DVP, ATRA, idarubicin therapy, Hoelzer chemotherapy regimen, La La chemotherapy regimen, ABVD, CEOP, 2-CdA, FLAG & IDA with or without concomitant G-CSF, VAD, Μ & P, weekly C, ABCM, MOPP and DHAP therapy.

The invention also provides methods for treating patients with B-cell lymphoma who are refractory to, among others, the above-listed treatments (i.e., treatment with anti-CD20 antibodies, bone marrow or stem cell transplantation, radiotherapy or chemotherapy). In particular, the invention provides methods for treating patients who do not show detectable tumor regression following administration of a chimeric anti-CD20 antibody with a radiolabeled anti-CD20 antibody.

In particular, the treatment of the patient with the radiolabeled anti-CD20 antibody is carried out after a period of about one week to two years following the treatment with the chimeric anti-CD20 antibody. More particularly, the treatment with the radiolabeled anti-CD20 antibody is carried out after a period of about one week to nine months following the treatment with the chimeric anti-CD20 antibody.

- 5 Although any anti-CD20 antibody may be used in the methods of the invention, the use of C2B8 (IDEG Pharmaceuticals, Rituximab®) is preferred. A preferred yttrium-90 ( ^90Y ) radiolabeled murine antibody is Y2B8. However, other antibodies containing a radioactive label, particularly β- or α-emitting isotopes, may also be used in the solution of the invention. Anti-CD19 antibodies may also be used.

The criteria for selecting a particular type of anti-CD20 antibody are known to those skilled in the art. For example, chimeric and humanized antibodies are advantageous because of their reduced immunogenicity and because they promote antibody-mediated immune responses through human constant region domains. In contrast, murine or other mammalian antibodies are advantageous for delivering radioactive label to tumor cells because they generally have a short half-life.

Initial antibody treatments that fail to improve or relapse patients may be with chimeric or mammalian antibodies. This also includes initial treatments with other antibodies, e.g. anti-CD19 and anti-Lym antibodies, as well as antibodies labeled with cytotoxic radicals, e.g. toxins and radioactive tracers [e.g. Oncolym® (Techniclone) or Bexxar (Coulter)].

The combined treatment regimens of the invention can be implemented by administering the aforementioned treatments simultaneously, i.e. the anti-CD20 antibody is administered at the same time or in the same period as the additional active ingredients (the treatments are administered side by side, but not at exactly the same time). The anti-CD20 antibodies of the invention can be administered before or after the additional treatments. In order to reduce the chance of temporary improvement or relapse, they can be administered regardless of whether the patient responds to the initial treatment or not.

The combination treatments of the invention include a method for treating B-cell lymphoma comprising administering at least one chimeric anti-CD20 antibody and at least one cytokine. More particularly, the invention provides a method for treating B-cell lymphoma comprising administering a synergistic therapeutic combination comprising at least one anti-CD20 antibody and at least one cytokine, the therapeutic effect of which is greater than that of the individual treatments combined. Preferred cytokines include interferon-α, interferon-γ, IL-2, GM-CSF and G-CSF. It is again emphasized that the anti-CD20 antibody and cytokine(s) may be administered in any order, sequentially or in combination.

The invention also provides a method for treating B-cell lymphoma, comprising administering to a patient a therapeutically effective dose of a chimeric anti-CD20 antibody prior to, concurrently with, or following a chemotherapy regimen. The chemotherapeutic

- 7 therapy regimens may be selected from a group consisting of at least the following: CHOP, ICE, mitosantrone, cytarabine, DVP, ATRA, idarubicin, Hoelzer chemotherapy regimen, La La chemotherapy regimen, ABVD, CEOP, 2-CdA, FLAG & IDA with or without concomitant G-CSF, VAD, Μ & P, weekly C, ABCM, MOPP and DHAP.

The invention also provides a method for treating B-cell lymphoma, comprising administering to a patient a therapeutically effective dose of a chimeric anti-CD20 antibody prior to, at the same time as, or following bone marrow or peripheral stem cell transplantation. Bone marrow transplantation may be used in conjunction with other treatment regimens, e.g. chemotherapy. The antibodies of the invention are also useful for reducing the number of CD20+ tumor cells remaining in the bone marrow or stem cells by administering chimeric anti-CD20 antibodies prior to or following myeloablative therapy. The antibodies may also be used in vitro to kill or reduce the number of remaining tumor cells in the bone marrow or stem cell preparations by inducing apoptosis of the tumor cells prior to returning them to the patient.

Stem cell transplants can be either allogeneic or autologous. In the case of allogeneic transplants, i.e. transplants from another person, the treatment regimens of the invention include treatments with immunosuppressive agents prior to the administration of anti-CD20 antibodies.

- 8. The invention also contemplates the simultaneous administration of other agents that promote transplant acceptance and the production and differentiation of immune cells. For example, it has been shown that GM-CSF treatment of bone marrow transplant patients promoted the development of specific bone marrow cells that produce circulating anti-infective neutrophil granulocytes, which improved their survival rate.

The methods of the invention are suitable for treating a variety of B-cell lymphomas, including low-grade/follicular non-Hodgkin lymphoma (NHL), small lymphocytic NHL, intermediate-grade/follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleft NHL, bulky disease NHL, and Waldenstrom's macroglobulinemia. It will be apparent to those skilled in the art that the names of the listed lymphomas often change with changes in classification systems, but the combined treatment regimens of the invention can be effectively used in the treatment of patients with lymphomas of various names.

For example, European and American pathologists have recently proposed a lymphoma classification system called REAL (Revised European American Lymphoma Classification). This system distinguishes mantle cell and marginal zone lymphomas from other peripheral B-cell neoplasms.

- 9-cell lymphomas and differentiates malignancy grades within certain classifications based on cytological examinations (e.g. small cell, mixed small and large cell, and large cell tumors). The combined treatment regimens according to the invention can also be successfully used in the treatment of all lymphomas of this classification.

The U. S. National Cancer Institute (NCI) has classified some of the REAL classes into more clinically applicable indolent or aggressive categories. Accordingly, indolent lymphomas include follicular cell lymphoma further classified according to cytological grade, diffuse small lymphocytic lymphoma/chronic lymphocytic leukemia (CLL), lymphoplasmocytoid/Waldenstrom's macroglobulinemia, marginal zone lymphoma, and spiny cell leukemia. Aggressive lymphomas include diffuse mixed and large cell lymphoma, Burkitt's lymphoma/diffuse small non-cleft lymphoma, lymphoblastic lymphoma, mantle cell lymphoma, and AIDS-associated lymphoma. The combination treatment regimens of the invention can also be used effectively in the treatment of these lymphomas.

Non-Hodgkin lymphomas are classified into low, intermediate, or high-grade categories based on the characteristics of the disease. Low-grade lymphomas are usually focal, indolent, or slow-growing. Intermediate- or high-grade lymphomas are generally more aggressive, characterized by the appearance of large, extranodal tumors. The combined treatment regimens of the invention are suitable for low-grade,

- 10 They can be used successfully in the treatment of both intermediate and high-grade NHLs.

The Ann Arbor staging system is also often used for patients with NHL. In this system, adult NHL cases classified as stage I, II, III, or IV are classified into categories B and A based on the presence or absence of certain well-defined general symptoms. Category B includes patients with the following symptoms: unexplained weight loss of more than 10% in the 6 months prior to diagnosis, fever of more than 38°C, and profuse night sweats. Occasionally, classification into the following stages is used.

Stage I: circumscribed involvement of a lymph node area or an extralymphatic organ or area.

Stage II: involvement of two or more lymph node regions on the same side of the compartment or circumscribed involvement of an extralymphatic organ or area and its regional lymph node, with or without involvement of other lymph node regions on the same side of the compartment.

Stage III: involvement of lymph node areas on both sides of the diaphragm, which may be accompanied by involvement of an extralymphatic organ or site, the spleen, or both.

Stage IV: disseminated (multifocal) process involving one or more extralymphatic sites with or without regional lymph node involvement, or extralymphatic

- 11 isolated process in a tic organ, accompanied by distant (non-regional) lymph node involvement. For further details, see: The International Non-Hodgkin's Lymphoma Prognostic Factor Project: A predictive model for aggressive non-Hodgkin's lymphoma. New England J. Med. 329(14): 987-994 (1993).

The following exemplary data are intended to illustrate preferred antibodies, dosage regimens, and specific combination treatments.

Rituximab® and Y2B8

Non-Hodgkin lymphoma affects approximately 250,000 people in the United States. The vast majority of NHL patients do not respond to chemotherapy or radiation therapy, or to high-dose autologous bone marrow (ABMT) or peripheral blood stem cell (PBSC) therapy.

Approximately 80% of NHLs are B-cell malignancies, more than 95% of which express the CD20 antigen on their cell surface. This antigen is a preferred target for immunotherapy because it is expressed only on B cells and not on hematopoietic stem cells, B-cell precursors, intact plasma cells, or other intact tissues. It is not shed from the cell surface and is not altered by antibody binding [Press et al.: Blood 69, 584 (1987)].

Rituximab® is a new generation of monoclonal antibodies created to overcome the disadvantages of murine antibodies (e.g. their short half-life, limited stimulation of human effector function, and immunogenicity).

- 12 members [Dillman: Journal of Clinical Oncology 12, 1497 (1994); Grossbard et al.: Blood 80, 863 (1992)].

Rituximab® is a genetically engineered monoclonal antibody that contains murine light and heavy chain variable regions and human gamma I heavy chain and kappa light chain constant regions. This chimeric antibody consists of two heavy chains of 451 amino acids each and two light chains of 213 amino acids each, with a molecular weight of approximately 145 kD. Rituximab® binds complement and mediates ADCC more efficiently than its murine parent molecule, and mediates CDC in the presence of human complement [Reff et al.: Blood 83, 435 (1994)]. This antibody inhibits the growth of FL-18, Ramos and Raji B-cell lines, sensitizes chemoresistant human lymphoma cell lines to diphtheria toxin, ricin, CDDP, doxorubicin and etoposide, and induces apoptosis in the human B-cell lymphoma cell line DHL-4 in a dose-dependent manner [Demidem et al.: Cancer Biotherapy & Radiopharmaceuticals 12, 177 (1997)]. The half-life of the antibody in humans is approximately 60 hours after the first infusion, which gradually increases to 174 hours after the fourth infusion. The immunogenicity of the antibody is low: only three (< 1%) of 355 patients tested in seven clinical trials had a detectable anti-chimeric antibody response (HACA).

Rituximab® is a genetically engineered derivative of the murine antibody 2B8. 2B8 is used in various diagnostic

- 13 has also been linked to various radioactive labels for diagnostic and therapeutic applications. To date, pending patent applications 08/475813, 08/475815 and 08/478967, which are incorporated herein by reference in their entirety, have described radiolabeled anti-CD20 conjugates for diagnostic imaging of B-cell lymphomas prior to administration of therapeutic antibody. The conjugate, In2B8, contains a murine monoclonal antibody specific for the human CD20 antigen, designated 2B8, linked to indium[III] (^In) with a bifunctional chelating agent, e.g., MXDTPA (diethylenetriaminepentaacetic acid). MXDTPA is a 1:1 mixture of l-isothiocyanatobenzyl-3-methyl-DTPA and l-methyl-3-isothiocyanatobenzyl-DTPA. The use of indium[111] as a diagnostic radionuclide is justified by its gamma-emitting properties and its use as an imaging agent.

Those skilled in the art are familiar with patent documents describing chelating agents and chelate conjugates. For example, Gansow, U.S. Patent No. 4,831,175, discloses polysubstituted diethylenetriaminepentaacetic acid chelates, protein conjugates containing them, and processes for their preparation. Gansow, U.S. Patent Nos. 5,099,069, 5,246,692, 5,286,850, and 5,124,471 also relate to polysubstituted DTPA chelates. All of these patents are incorporated herein by reference in their entirety.

- 14 The choice was made for the specific bifunctional chelating agent MX-DTPA because it has an increased affinity for trivalent metals, ensures a higher tumor:intact tissue ratio, has low bone incorporation and allows for increased retention of the radionuclide in the target tissue in vitro, e.g. in B-cell lymphomas. However, other bifunctional chelating agents are also known to the skilled person, which can also be advantageously used during tumor therapy.

United States Patent No. 5,736,137 also discloses radiolabeled therapeutic antibodies for the targeting and destruction of B-cell lymphomas and tumor cells. Specifically, the conjugate Y2B8 contains the same murine anti-human CD20 monoclonal antibody 2B8 bound to yttrium[90] ( ⁹⁰ Y) with the same chelating agent as above. The therapeutic use of this radionuclide is justified by its several favorable properties. The half-life of 64 hours of ⁹⁰ Y is long enough for the antibody to accumulate in tumors and, unlike ¹³¹ I, it emits pure high-energy β-radiation with a range of 100-1000 cell diameters, and does not produce concomitant gamma radiation during its decay. The minimal amount of penetrating radiation allows the use of ⁹⁰ Y-labeled antibodies in outpatient treatment. In addition, the uptake of the labeled antibodies is not necessary for cell destruction, and the locally emitted ionizing radiation is also fatal to adjacent tumor cells that do not contain the target antigen.

- 15 Since the ⁹⁰ Y radionuclide was bound to the 2Β8 antibody by the MX-DTPA bifunctional chelating molecule, the Y2B8 molecule also has the above-described advantageous properties, such as e.g. increased radionuclide retention observed at the target site. However, unlike ^nl In, it cannot be used for imaging, as it does not emit gamma radiation. Based on all of this, in the combined treatment protocols of the invention, the location and relative size of a tumor can be determined with a radionuclide suitable for diagnostic imaging, e.g. ^In, before and/or after the administration of the therapeutic chimeric or ⁹⁰ Y-labeled antibodies. The use of the indium-labeled antibody also provides the possibility of dosimetric estimation.

Depending on the desired (diagnostic or therapeutic) use of the antibody, the use of other radioactive labels is also known to the skilled person. Radiolabels used in clinical diagnostics include ¹³¹ I, ¹²⁵ I, ¹²³ I, ⁹⁹ Tc, ⁶⁷ Ga and ¹ In. Several radioactively labeled antibodies may also be potentially used in targeted immunotherapy [Peirersz et al.: Immunol. Cell Biol. 65, 111 (1987)]. Such radionuclides include ¹⁸⁸ Re, ¹⁸⁶ Re and ⁹⁰ Y and to a lesser extent ¹⁹⁹ Au and ⁶⁷ Cu. I-[131] is also used for therapeutic purposes. United States Patent No. 5,460,785, which is incorporated herein by reference in its entirety, lists such radioactive isotopes.

- 16 According to United States Patent No. 5,736,137, the administration of a radiolabeled Y2B8 conjugate and an unlabeled chimeric anti-CD20 antibody to mice bearing B-cell lymphoblastic tumors resulted in significant tumor regression. In addition, in human clinical trials described in the same publication, a significant reduction in B-cell numbers was observed in lymphoma patients who received chimeric anti-CD20 antibody infusions. Chimeric 2B8 was recently announced as the first FDA-approved antitumor monoclonal antibody available in the United States under the name Rituxan®. Based on these findings, at least one chimeric anti-CD20 antibody has been shown to be effective in the treatment of B-cell lymphoma.

U.S. Patent No. 5,736,137, which is part of the teaching, also reported the sequential administration of the chimeric anti-CD20 Rituxan® and indium- or yttrium-labeled, or both, murine monoclonal antibodies. Although the radiolabeled antibodies used in this combination therapy are murine, the depletion of B cells due to the initial treatment with chimeric anti-CD20 results in a weaker ΗΑΜΑ response, which facilitates the use of a combination therapeutic or diagnostic treatment regimen.

In the context of combination immunotherapy, murine antibodies can therefore be used as diagnostic reagents. In addition, U.S. Patent No. 5,736,137

- Patent document 17 shows that a therapeutically effective dose of yttrium-labeled anti-CD20 antibody administered following Rituxan® treatment is capable of (a) eliminating all B cells in the peripheral blood that are not killed by the chimeric anti-CD20 antibody; (b) initiating B cell depletion in lymph nodes or (c) other tissues.

Therefore, the combination of anti-tumor antibodies with radioactive tracers represents a valuable clinical tool for evaluating the potential therapeutic effect of such antibodies, for developing diagnostic reagents for monitoring the efficacy of treatment, and for developing additional therapeutic reagents that enhance the tumor cell killing effect of the chimeric antibody. Based on the proven efficacy of the anti-CD20 antibody in the treatment of non-Hodgkin lymphoma and the well-known sensitivity of lymphocytes to radioactivity, the use of such chimeric and radioactively labeled therapeutic antibodies in therapeutic regimens that reduce the incidence of relapsed or refractory non-Hodgkin lymphoma would be of great benefit. In addition, it would be advantageous if these combined regimens were also used in the treatment of other B-cell lymphomas.

- 18 .....· ··:· ·-· ·

Low-grade or follicular NHL

Single-agent trials for the treatment of relapsed or refractory NHL

The FDA approval of Rituximab® was based primarily on five single-agent trials in patients with low-grade or follicular NHL. In an early phase I trial involving single infusions of Rituximab® at doses of 10 to 500 mg/ ^m2, the maximum tolerated dose was not achieved; however, the duration of infusion at the highest dose did not allow for outpatient use. The ORR in 15 patients was 13% (Table 1) [Maloney et al.: Blood 84, 2457 (1994)].

Ά Summary of Rituximab® efficacy studies

• • · · · • ·« ;· ·« Ref e- rench; 44 l> 21,22 cn co Median TIP (month) 1 '8 (M O 36, 7 + 13.2 Median 1 DR (snow- day) NA 8, 6 35.3+ 11, 6 PR i CM o\o CO 14 o\o 16 o\° CN o\o CR O o\o O <n o\° ΟΊ i 22 (58%) σ> o\o NOSE CM o\° 00 17 (50%) _ 38 (100%) 76 (50%) 15 34 CO 00 151 Indication relapsed B-cell lymphoma renewable low-, medium- and high grade of malignancy lymphoma you are newly diagnosed recurrent low-grade malignancy degree or follicular B- cellular lymphoma 1 recurrent low-grade malignancy degree or follicular B- cellular lymphoma Experiment Description Phase I/II. single dose, one active ingredient Phase I/II, multi-dose, dose- adjuster Phase II. Combined with CHOP- said Phase III, multiple doses, one active ingredient

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♦ ·* · ·**· · ** References 19.20 *· * • · • ♦ 1 • t'j ί............................. Median TIP (month) + kO Median DR (months) 15, 0+ PR 17 (29%) CR o\O kO r4 NOSE 23 (40%) * 57 Indication relapsed low-grade or follicular B-cell lymphoma, retreatment Experiment Description Phase II, multi-dose, single-agent

34 33 + — 2 + co + rH the o\o , CO CN co 19 (63%) f 29 (96% 17 (32% 1 o 00 HORSE OJ 1 muscular grade as pronounced .3 ύη ω <0 1 'Η β m d G rö >i 'p tö 2 years old tn Λ4 g -Η Ο (ΰ Ο Ό $ H q-l > Mon-Fri Φ β β ci β Ή I ·Η Ή β ω ω —i W Φ '(Ü ^{Mr. K} Λ -Ρ <Ű <- ⁰¹ φ Ή β 5 ν ρ 'p Φ p; Λ * :Ο tn Ai -Η s 2 ⁰¹ (ϋ Ai β Φ n O s Λί tn Φ -M Λ Ή 1 Φ , more OP-changed- ί, plural s Λ +J rd -P Ti Η Φ Φ phase os, CH jinal zat phase :native dose THE! tendon 'Φ p - P Ö P - H 'Φ £ tn h p 2 η <ΰ II. tei II II s < * a

- 22 In the first phase of the phase I/II dose-titration trial, patients received 125-375 mg/m ² of the active substance as a weekly infusion, for a total of four times. No dose-related toxic effects were observed, so the dose of 375 mg/m ² was used in phase II. Of the 37 patients treated with this dose, 17 (46%) had tumor regression, which was complete response (CR) in 3 (8%) patients and partial response (PR) in 14 (38%) patients [Maloney et al.: Blood 90, 2188 (1997)].

Subsequently, a single-arm, pivotal trial was conducted in which 166 patients with relapsed or refractory, low-grade or follicular NHL (International Working Formulation (IWF) type ^AD , REAL classification: small lymphocytic lymphoma, follicular center lymphoma, follicular lymphoma grade I, II, III) were given 375 mg/m2 of Rituximab® four times a week [McLaughlin et al.: Journal of Clinical Oncology 16, 2825 (1998)]. Patients with tumors larger than 10 cm or with more than 5000 lymphocytes per μl in their peripheral blood were excluded from the study. The median age of the patients (105 men and 61 women) was 58 years, and the median number of prior therapies was 3. Of the 149 patients evaluated, 56% had bone marrow involvement. 45% had 2 or more extranodal nodules, and 41% had disease with extensive (> 5 cm) tumor formation.

- 23 The criteria for complete remission were as follows: regression of all lymph nodes to less than 1x1 ^cm2 on two CT scans of the neck, chest, abdomen, and pelvis, at least 28 days apart, resolution of all lymphomatous signs and symptoms, and resolution of bone marrow, liver, and spleen lesions. Partial remission was defined as a reduction of at least 50% in the product of the perpendicular dimensions of the lesions and no evidence of disease progression for at least 28 days. Patients who did not achieve CR or PR were considered non-responders, even if their overall measurable disease characteristics improved (>50%). Time to disease progression was calculated from the first infusion to the time of disease progression.

The overall response rate (ORR) was 48%, with 6% CR and 42% PR [McLaughlin et al.: Journal of Clinical Oncology 16, 2825 (1998)]. The median time to progression (TPP) and the median duration of response (DR) in responding patients were 13.2 months and 11.6 months, respectively. Of the 80 responding patients, 22 (28%) remained symptom-free for 20.9 to 32.9 months [McLaughlin et al.: Blood 92, 414a (1998)].

Rituximab® administration resulted in a rapid and prolonged reduction in the number of circulating B cells. The number of circulating B cells was established within the first three doses.

- 24 The reduction in culture was maintained in 83% of patients for 6-9 months after treatment. After treatment, the mean B-cell count returned to physiological levels. Although the mean natural killer (NK) cell count did not change, a positive correlation was observed between the effectiveness of Rituximab® therapy and higher baseline NK cell counts [Janakiraman et al.: Blood 92 (10 suppl. 1.), 337a (1998)].

Several key prognostic factors were examined to determine their association with improvement. In the 23 patients who relapsed after ABMT or PBSC, the ORR was 78%, compared with a significantly lower 43% in patients who had not received prior high-dose therapy (p < 0.01). Multivariate analysis showed that the ORR was significantly higher in patients with follicular NHL than in patients with small lymphocytic lymphoma (58% vs. 12%, p < 0.01), as was the case when comparing patients with chemosensitive and chemoresistant relapse (53% vs. 36%, p < 0.06). The improvement rate was not affected by age > 60 years, the presence of extranodal disease, prior anthracycline therapy, or bone marrow involvement.

A statistically significant correlation was observed between the mean serum antibody concentration and improvement with multiple measurements during treatment and follow-up [Berinstein et al.: Annals of Oncology 9, 995 (1998)].

- 25 Patients with follicular NHL had higher serum antibody concentrations than those with small lymphocytic lymphoma. The mean serum antibody levels were inversely related to tumor size and baseline circulating B-cell counts. The association between lower serum antibody concentrations and higher circulating NHL cell counts and larger tumor size suggests that antibody clearance is associated with tumor cells. The improvement observed with high serum antibody concentrations, smaller tumor size, and fewer circulating cells suggests that higher doses or multiple courses of Rituximab® may be required to achieve improvement in certain patient groups, such as patients with extensive tumor growth.

In addition, 43% of patients with tumors larger than 5 cm and 35% with tumors larger than 7 cm treated with Rituximab® experienced improvement, indicating the potential of Rituximab® in the treatment of bulky tumors. This is surprising given that antibody therapy was previously considered unsuitable for the treatment of bulky tumors due to the dense nature of the tumor growth.

In a trial conducted in Japan, patients with relapsed B-cell lymphoma were treated with Rituximab® at a dose of 250 mg/ ^m2 (N = 4) or 375 mg/m2 (N = 8) weekly for a total of ^four sessions [Tobinai et al.:

- 26 Annals of Oncology 9, 527 (1998) ] . Of the 11 evaluable patients, 8 had follicular NHL, 2 had diffuse large cell NHL, and 1 had mantle cell lymphoma. The ORR for the group was 64%, with 2 patients achieving complete remission and 5 achieving partial remission, all of whom had follicular-type lesions.

Since previous studies have shown a positive correlation between serum Rituximab® concentrations and response, we initiated a phase II study of 375 mg/ ^m2 weekly Rituximab® for 8 weeks in patients with low-grade or follicular NHL. The ORR for evaluable patients was 60%, with 14% CR and 46% PR. In patients who responded, the median TTP was >13.4 months and the median DR was >19.4 months [Piro et al.: submitted for publication (1999)]. Although it is difficult to compare results from different trials, it appears that TTP and DR can be improved by increasing the dose.

Contrary to previous assumptions that antibody therapy is only suitable for the treatment of processes involving micro-metastasis, Rituximab® has also been shown to be quite effective in the case of disease involving bulky tumor tissue formation. In a further trial, 31 patients with low-grade, relapsed or therapy-resistant NHL with bulky tumor tissue formation (> 10 cm in diameter, discrete) were treated with Rituximab® at a weekly dose of 375 mg/m ² for four weeks. Of the 28 evaluable patients, 12 (43%) had a response.

- CR (1 patient, i.e. 4%) or PR (11 patients, i.e. 39%) were observed in 27 cases [Davis et al.: Blood 92 (10 suppl. 1.), 414a (1998)].

Waldenstrom's macroglobulinemia

Waldenstrom's macroglobulinemia (WM) is a malignant tumor in which the tumor B lymphocytes produce abnormally high levels of IgM antibodies. WM usually occurs in patients over the age of sixty, but has been described in adults in their early thirties. WM is currently considered a rare, incurable, indolent tumor type, and plasmapheresis was previously used to reduce serum viscosity. Chemotherapy is usually recommended with an alkylating agent and corticosteroids. The most commonly used compound for the treatment of WM is Leustatin (2CdA).

In a study of seven patients with Waldenstrom's macroglobulinemia treated with 375 mg/ ^m2 of Rituximab® once weekly for four weeks, 4 (57%) showed improvement [Byrd et al.: Blood 92 (10 suppl. 1.), 106a (1998)]. The median progression-free survival was 8 months (range: 3-27+ months). Accordingly, Rituximab® may be suitable for use in combination treatment protocols with chemotherapeutic agents, e.g. 2CdA.

Chronic lymphocytic leukemia (CLL)

CLL is the liquid (leukemic) counterpart of small lymphocytic lymphoma. It is treated with a standard dose of Rituximab®.

- 28 patients with SLL had lower serum concentrations and improvement rates compared to other low-grade NHL subtypes. This is likely due to the extremely high circulating tumor cell concentration in CLL and the fact that malignant tumor cells in CLL are known to have reduced cell surface CD20 expression.

We have also shown that Rituximab® can be used for the treatment of hematological malignancies, e.g. CLL. A recent clinical trial evaluated the effect of high-dose Rituximab® treatment in CLL patients [O'Brien et al.: Blood 92, 105a #431 (1998)]. In order to minimize post-infusion side effects, each patient received an initial dose of 375 mg/m ³ . Subsequently, the drug was titrated three times weekly, and 16 patients were treated with doses of 500-1500 mg/m ³ . The median age of the patients was 66 years (range: 25-78 years). 81% of the patients had terminal, stage III-IV disease. Their mean white blood cell count was 40 x 10 ⁹ /L (range: 4-200), their hemoglobin concentration was 11.6 g/dl (range: 7.7-14.7), their platelet count was 75 x 10 ⁹ /L (range: 16-160), and their mean B ₂ immunoglobulin concentration was 4.5 mg/L (range: 3.1-9.2). The mean number of previous treatments was 2.5 (range: 1-9). 60% of the patients did not respond to treatment. Two patients developed a severe increase in blood pressure after the first treatment (375 mg/m ³ ), and one additional patient

- 29 patients received additional treatment. The successively increased doses caused only mild toxicity, although we could not evaluate the data of the patients who received the 1500 mg/m ³ dose in any case. We completed the treatment in eight patients (four completed at 500 mg/m ³ , three at 650 mg/m ³ , and one at 825 mg/m ³ ). One patient treated with a dose of 560 mg/m ³ achieved complete recovery. One patient is receiving treatment against progressive lymphocytosis, the other patients had a decrease in peripheral lymphocytosis, but the treatment had less effect on their lymph nodes. Our experiments with increasing the therapeutic drug dose are currently ongoing.

Another approach is to further improve the efficacy of therapy in CLL patients by increasing CD20 antigen expression with cytokines. In an in vitro study, mononuclear cells from CLL patients were incubated with various cytokines for 24 hours. Flow cytometric studies showed that IL-4, GM-CSF and TNF-α resulted in a significant increase in expression [Venugopal et al.: Blood 10, 247a (1998)]. Furthermore, some recent experimental results suggest that the increase in CD20 expression observed in CLL cells is limited to tumor cells [Venugopal et al.: Cytokine-induced upregulation of CD20 antigen expression in chronic lymphocytic leukemia (CLL) cells may be limited to tumor cells poster, PanPacific Lymphoma Meeting (1999)]. Based on preliminary experimental results, α-interferon at concentrations of 500-1000 U/ml

- In 30 cases, it increases CD20 expression on CLL cells after just 24 hours.

Based on these findings, the expression of CD20 on the surface of malignant B cells can be increased by administering certain cytokines to CLL patients before or at the same time as their Rituximab® treatment, which makes CD20 and other cell surface markers, e.g. CD19, a more advantageous target for immunotherapy. We have established an experimental collaboration to determine the optimal cytokine doses for increasing CD20 expression in vivo. At the beginning of the experimental protocol, 10 patients are treated with 250 mcg/m ² MG-CSF, 10 patients with mcg/kg IL-4, and 10 patients with 5 mcg/kg GCSF, SQ QD X 3. To determine whether the increase in CD20 expression results in increased tumor cell death caused by Rituximab® treatment, apoptosis studies are performed. For this purpose, mononuclear cells are obtained from patients by Ficon Hypaque centrifugation.

Antibody therapy for CLL can be combined with other conventional chemotherapeutic regimens known to be effective against CLL. The most commonly used compound alone for this purpose is chlorambucil (Leukeran), which is administered either at a dose of 0.1 mg/kg daily or at a dose of 0.4-1.0 mg/kg every four weeks. Chlorambucil is often combined with oral prednisolone (30-100 mg/ ^m2 /day), which is advantageous in the treatment of autoimmune cytopenias. A possible alternative to chlorambucil is cyclophosphamide, which is typically administered at a dose of 1-2 g/ ^m2 every 3-4 weeks, with vincristine and

- 31 are used in combination with steroids (e.g. COP treatment regimen).

Several drug combinations are used for the treatment of CLL, such as COP (cyclophosphamide, Oncovin and prednisolone) and CHOP (the previous three drugs plus doxorubicin). Fludarabine has shown some efficacy in the treatment of CLL, with a 50% ORR achieved in one patient group at a dose of 25-30 mg/m ² /day every 3-4 weeks. However, some patients did not respond to fludarabine treatment. These patients may also respond similarly to 2-CdA therapy, since diseases resistant to fludarabine are usually resistant to 2-CDA treatment [O'Brien et al.: N. Engl. J. Med. 330, 319 (1994)].

Based on all these, anti-CD20 antibody therapy is particularly beneficial for patients who have not responded to treatment with chemotherapeutic agents or who have subsequently relapsed. In their case, Rituximab® treatment can also be combined with radiotherapy. Total body irradiation (TBI) with a low single dose (15 cGy), applied in a total dose of 75-150 cGy, has proven effective in about a third of patients.

A phase II study of CLL patients, conducted by CALGB, is currently underway. In one group, Rituximab® and fludarabine are given simultaneously, followed by Rituximab® treatment, and in another, fludarabine induction is followed by Rituximab® therapy.

- 32 * ······ ·· « ······· · ·*

Combined use of Rituximab® and myeloablative therapy

Myeloablative therapy has been successful in indolent lymphomas, although tumor cells may remain despite high-dose therapy, and the returned PBSC may contain tumor cells. Rituximab® is used before stem cell removal and after transplantation to reduce the number of residual CD20+ tumor cells and to avoid contamination of the recovered bone marrow or stem cells. Preliminary data suggest that CD20+ cells were not among the recovered cells. Eighteen of 24 patients underwent transplantation and tolerated the treatment well. PCR studies to detect residual tumor cells are currently underway [Flinn et al.: Blood 92, 648a #2673 (1998)].

Recurrent low-grade NHL with repeated Rituximab® treatment

In one study, the effect of retreatment of 53 patients with cancer who had initially responded to Rituximab® but subsequently relapsed was investigated [Davis, T.: Blood 92 (10 suppl. 1), 414a (1998)]. Of the 56 evaluable patients, 7 (13%) had a CR and 16 (29%) had a PR (42% ORR). Four patients who responded to the second treatment received a third treatment, and three of these showed improvement.

After two treatments with Rituximab®, a patient's tumor previously diagnosed as follicular, small cleft cell NHL no longer expressed the CD20 antigen and, along with its transformation into diffuse, large cell NHL, no longer responded to Rituximab® treatment [Davis et al.: Clinical Cancer Research, forthcoming (1999)].

Accordingly, while repeated Rituximab® treatment is effective in patients with tumor recurrence following prior Rituximab® therapy, the incidence of CD20- tumor cells may increase following the second treatment. This observation also supports the possibility of using the combined treatment regimens of the invention.

Treatment of low-grade NHL with the combination of Rituximab® and CHOP chemotherapy

Chemotherapy with cyclophosphamide, doxorubicin, vincristine and prednisolone (CHOP) is an effective first-line treatment for low-grade or follicular NHL. Although the initial response rate is high, the tumor relapses later and subsequent chemotherapy regimens provide only shorter remission periods. A phase II study investigated the effect of CHOP chemotherapy in combination with Rituximab® in newly diagnosed and relapsed low-grade or follicular NHL [Czuczman et al.: Journal of Clinical Oncology 17, 268 (1999)]. Due to the different mechanisms of action of the two regimens, cross-resistance is unlikely, and synergism can be observed when Rituximab® is administered with certain cytotoxic agents, e.g. doxorubicin.

- 34 * ···»·· · ·

[Demidem et al.: Cancer Biotherapy and Radiopharmaceuticals 12, 177 (1997)].

Of the 38 patients, 29 had not received prior anticancer therapy. CHOP treatment was performed at a standard dosage every three weeks for six cycles, with six Rituximab® infusions (375 mg/m ² ). Rituximab® infusions 1 and 2 were administered on days 1 and 6 prior to the first CHOP cycle, which started on day 8. Rituximab® infusions 3 and 4 were administered two days prior to CHOP cycles 3 and 5, and infusions 5 and 6 were administered 134 and 141 days after CHOP cycle 6.

In this study using combination therapy, all 38 patients showed improvement (58% CR, 42% PR). Of the 35 patients who completed treatment, 63% had CR and 37% had PR [Czuczman et al.: Journal of Clinical Oncology 17, 268 (1999)]. The median DR was over 35.3 months, and the median relapse-free survival could not be determined over the observation period of 36.7 months. 22 of the patients had been symptom-free for more than 36 to 53.4 months [White et al.: Proceedings of ASCO, forthcoming (1999)]. This DR value would be noteworthy even in the case of primary treatment, where 24% of the patient population had relapsed after prior chemotherapy.

In a planned CALGB trial, 40 patients with low-grade NHL will receive Rituximab® once weekly for eight weeks and oral cyclophosphamide once daily from day eight. Twenty patients will receive only

- 35 • ······ ·· « ···*♦<· * «Μ

You will receive Rituximab® treatment once a week for eight weeks.

A phase III study conducted by ECOG is comparing the effects of cyclophosphamide and fludarabine (group A) and standard CVP therapy (group B) in patients with low-grade malignancy. Patients are randomly assigned to group A or B based on stratification based on age, tumor stage, histopathology, and B symptoms. Patients in both groups who show improvement undergo a second stratification to receive maintenance Rituximab® therapy (group C, 375 mg/ ^m2 four times a week every six months for two years) or observation only (group D).

Combined use of Rituximab® and cytokines

Combined use of Rituximab® and interferon-α

Interferon is a cytokine that influences the function of the immune system [Wadler et al.: Seminars in Oncology 19, 45 (1992)]. Interferon can increase the effectiveness of antibodies by promoting antigen expression [Nakamura et al.: Oncology 50, 35 (1993)], by enhancing the targeting of antibodies to tumor tissue [Greiner et al.: Science 235, 895 (1987); Murray et al.: Journal of Biological Response Modifiers 9, 556 (1990)], and by enhancing the cytotoxicity of immunotoxins [Yokota et al.: Cancer Research 50, 32 (1990)].

In a combination treatment trial, patients with relapsed low-grade or follicular NHL were

- 36 .··.....· r

In NHL, a clinically active cytokine [Grillo-López et al.: Antibody Immunoconjugates and Radiopharmaceuticals 8^, 60 (1995)] was administered along with α-interferon (RoferonA) and Rituximab®. α-interferon (2.5 or 5 MIU subcutaneously) was administered three times a week for 12 weeks. Rituximab® was administered as an intravenous infusion of 375 mg/m ² four times a week starting from the fifth week of treatment. The ORR was 45% (17 of 38 patients), with 11% of patients achieving a CR and 34% achieving a PR. For patients who improved, the Kaplan-Meier estimates of median DR and TPP were over 22.3 and 25.2 months, respectively [Davis et al.: Proceedings of the American Society of Clinical Oncology 17, 11a (1998)]. In previous studies of the combination of α-interferon and anthracycline-containing regimens, although the time to disease progression was increased, the survival or improvement rate was not significantly changed [Smalley et al.: New England Journal of Medicine 327, 1336 (1992); Hagenbeek et al.: Journal of Clinical Oncology 16, 41 (1998); SolalCéligny et al.: Journal of Clinical Oncology 16, 2332 (1998)]. These preliminary results suggest that the combination of α-interferon and Rituximab® may prolong the time to disease progression compared to Rituximab® alone.

Combined use of Rituximab® and G-CSF

In a further ongoing trial, the combined use of Rituximab® and G-CSF has been shown to be effective in the treatment of recurrent,

- 37 We are investigating the effect of G-CSF on low-grade NHL. It has been established both in vitro and in vivo in healthy volunteers that G-CSF induces the formation of FcRI-positive neutrophil granulocytes, which function as effector cells in ADCC, through its effect on myeloid precursor cells. Based on this, we have initiated a phase I/II trial to investigate the toxicity and efficacy of the combination treatment.

In both Phase I and II, patients were treated with a standard dose of G-CSF (5 μg/kg/day) starting two days before the start of Rituximab® treatment for three days. Phase I involved escalating the Rituximab® dose (125, 250 or 375 mg/m ² , four times, once a week). Based on the data of the 9 patients evaluated so far, the ORR was 67% (CR 44%, PR 22%), with minor toxic side effects observed in eight patients [van dér Kőik et al.: Blood 92, 241b #4037 (1998)]. The most common side effects were fever (in four of eight patients), rhinitis (4/8), chills (3/8) and headache (3/8), which correspond to those previously observed with Rituximab® alone. The trial has already started Phase II. Phase 1 of the trial aims to determine the efficacy of the combination of G-CSF and four doses of Rituximab® at 375 mg/m ² .

Combined use of Rituximab® and IL-2

High-dose autologous peripheral blood stem cells (PBSC) or surviving bone marrow (BM) therapy has also been used to treat NHL, but the success of this treatment is limited, with a high risk of disease recurrence ⁽ 50-80%). In order to achieve long-term remission after transplantation, immunotherapy using high or low doses of IL-2 has been performed in several institutions. The results of these indicate that IL-2 therapy is indeed effective against tumors in the early period after transplantation.

After autologous transplantation, recovery of the immune response in patients is delayed, which may result in a decrease in the degree of immune-mediated tumor killing [Witherspoon et al.: Semin. Hematol. 21, 2 (1984); Anderson et al.: Blood 69, 597 (1987)]. Indeed, it has been shown that both CD8+ and cytotoxic CD8+ T-cell numbers are low [Lum: Blood 69, 369 (1987); Azogui et al.: J. Immunol. 131, 1205 (1983); Welte et al.: Blood 64, 380 (1984); Cayeau et al.: Blood 7 4, 2270 (1989); Bosley et al.: Nouv. Rev. Fr. Hematol. 32, 13 (1990)]. In vitro studies have shown a significant suppression of T-cell cytolytic and proliferative responses and a decrease in IL-2 production in response to mitogens and soluble antigens. However, soluble IL-2 is able to restore these immune response processes, as shown by the ability of immune cells of autologous transplant patients to respond to exogenous IL-2 [Welte et al.: Blood 64, 380 (1984)]. After bone marrow transplantation, peripheral blood natural killer activity also remains below control values, which is also corrected by the addition of IL-2 [Bosley et al.: Nouv. Rév. Fr. Hematol. 32, 13 (1990)].

- 39 *- ··*· 1 φ· « « « · « « · · • ·· 4 «α ♦·· · < · :* ' * ··

All of this suggests that IL-2 given shortly after stem cell transplantation may enhance immune responsiveness during the critical period characterized by minimal tumor tissue resistance and immunosuppression due to IL-2 deficiency.

Caligiuri et al., for example, showed that 0.45 x ¹⁰⁶ U/ ^m2 /day of IL-2 (Hoffman-LaRoche) administered 24 hours a day for 12 weeks was able to increase the number of CD56+ NK cells [Caligiuri et al.: J. Clin. Oncol. 9, 2110 (1991); Caligiuri et al.: J. Clin. Invest. 91, 123 (1993); Caligiuri: SEM in Oncol. 20, 3 (1993)]. This could be used in the outpatient treatment of patients who did not receive the treatment-prescribed transplant with little toxicity.

In animal models, it has been shown that low doses of non-LAK-inducing IL-2, when given together with tumor-specific effector T cells, significantly enhanced the antitumor effect [Klarnet et al.: J. Immunol. 138, 4012 (1987)]. Soiffer et al. 13 bone marrow transplant recipients with reduced autologous bone marrow or T-cell counts undergoing treatment for relapsed leukemia or lymphoma were treated with low doses of IL-2 [Soiffer et al.: Blood 79, 517 (1992)]. Laboratory results indicated an enhanced immune response, as indicated by a 540-fold increase in the number of circulating CD56+, CD16+, and CD3- NK cells. In addition, this low-dose IL-2 therapeutic regimen resulted in enhanced in vitro killing of K562 NK target cells. 29

- 40 - .- .w. . .• ♦ * * « ·** *w In evaluating the outcome of treatment in allogeneic bone marrow transplant recipients, Soiffer et al. showed that these patients had a higher survival rate compared to controls with the same histological diagnosis (70% versus 30%, p = 0.41) [Soiffer et al.: Blood 84, 964 (1994)].

Lauria et al. 11 patients with high-grade NHL were treated with 2 x 10 ⁶ IU/m ² IL-2 four times daily for two weeks after autologous bone marrow transplantation, followed by 3 x 10 ⁶ IU/m ² twice weekly for one year [Lauria et al.: BMT 18, 79 (1996)]. Phenotypic analysis 6 months after treatment indicated a significant (p = 0.001) and sustained increase in the proportion and absolute number of lymphocytes, especially in the case of CD6+ and CD56+ NK cells. No deterioration was observed in any patient 22 months after initial therapy (range: 10-42 months). In addition, the liver and lymph node lesions remaining in two patients after autologous bone marrow transplantation healed without a trace after 7 and 10 months of IL-2 treatment.

Vey et al. treated patients with refractory or relapsed HD (11 cases) and NHL (14 cases) with low-dose IL-2 [Vey et al.: Leukemia & Lymphoma 21, 107 (1996)]. In 48% of the patients, the disease was refractory at the time of autologous bone marrow transplantation, and CR was observed in 84% of them. The average time between transplantation and the start of IL-2 treatment was 54 days, and the treatment consisted of a first 5-day cycle, followed by 4 additional 2-day cycles every 2 weeks. The patients received an average of 160 x 10 ⁶ IU/m ² of IL-2. Based on five-year follow-up data, the survival and disease-free survival (DFS) probabilities are 72% (HD: 73%, NHL: 70%) and 45% (HD: 36%, NHL: 48%) respectively.

A team at the Fred Hutchinson Cancer Research Center (FHCRC) recently found that low-dose IL-2 therapy was well tolerated in an outpatient setting and resulted in longer periods of remission compared with no treatment. IL-2 therapy resulted in increases in certain immune cell subsets, including CD8+ and CD69+, CD16+ and CD8+, CD16+ and CD69+, CD16+ and CD56+, CD16+ and CD122+, CD16+ and Dr+, and CD8+ and CD56+ cells. Median increases in lytic activity against the tumor targets K562 and Daudi of 5.9-fold and 6.5-fold, respectively, were also observed. The median time from transplant to possible disease recurrence was 17.8 months, a much longer improvement than previously observed in transplant recipients who did not receive IL-2 treatment.

Based on the favorable experience of patients receiving autologous bone marrow transplantation treated with IL-2 alone, it seemed logical to combine IL-2 treatment with Rituximab® therapy after transplantation, as the biological effect of the latter is presumably exerted through ADCC and complement-mediated lytic activity.

- 42 nyesül. Accordingly, we have initiated a phase I study in collaboration with the FHCRC to determine the safety and potential efficacy of the aforementioned combined treatment regimen.

Another phase II study is to determine the efficacy and incidence of HACA formation in patients receiving low-dose IL-2 and Rituxan®. One of the objectives of this study is to determine whether the presence of IL-2 in vivo enhances ADCC and whether ADCC activity is associated with clinical response to therapy. Patients were eligible for the trial if they had a histopathological diagnosis of stage II-IV, low-grade follicular B-cell or mantle cell lymphoma. For the purposes of this clinical study, a tumor that was CD5+, CD23- (if applicable), and/or bcl-1+ based on immunohistochemistry was considered mantle cell lymphoma. Patients who have not responded to or relapsed after standard treatment (e.g. chemotherapy, radiotherapy, autologous bone marrow transplantation and/or immunotherapy) may also be included in the study.

Combined use of Rituximab® and GM-CSF in the treatment of relapsed, low-grade or follicular B-cell lymphoma

We have initiated two additional separate Phase II studies to evaluate the efficacy of the combination of Rituximab® and GM-CSF. One of the studies was conducted in 40 patients with relapsed, low-grade

- It involves 43 patients with advanced B-cell lymphoma and consists of 375 mg/m2 ^of Rituximab® administered subcutaneously once a week for a total of four times (on days 1, 8, 15 and 22) and 250 μg of GM-CSF (Leukine, Immunex) administered subcutaneously three times a week for eight weeks, one hour before the first dose of Rituximab®. This study aims to evaluate the clinical efficacy [ORR; overall complete response rate (OCRR); TTP; failure free survival)], safety (quantity, quality, duration and reversibility of side effects) of the combination regimen, as well as its effect on the lymphocyte subsets and cytokines of interest. During the second study, immunological parameters are monitored to clarify the mechanism of cell destruction (C3 and C4 complement determination, flow cytometry for determining CD3, CD4, CD8, CD16, CD19 and CD56, ADCC assay).

Combined use of Rituximab® and γ-interferon

In the treatment of low- and high-grade lymphomas, Rituximab® treatment can be usefully supplemented with γinterferon therapy. It has recently been shown that γinterferon increases CD20 expression on plasma cells, B cells from multiple myeloma (MM) patients, and B cells from healthy donors [Treon et al.: Lugano (1999)]. Treon et al. also showed that γ-interferon promotes the binding of these cells to Rituximab®. The plasma cells express CD20

- 44 expression was induced in a dose-dependent manner, with an increase in expression observed at a concentration of 1 U/ml γ-interferon. The expression curve reached a plateau within 48 hours at a dose of 100 U/ml. Based on all of this, the co-administration of γ-interferon with Rituximab® may also be beneficial.

Intermediate- and high-grade non-Hodgkin's lymphoma

Single-agent studies

A study conducted in Europe and Australia evaluated alternative dosing protocols in 54 patients with relapsed or refractory intermediate- or high-grade NHL [Coiffier et al.: Blood 92, 1927 (1998)]. Rituximab® was administered weekly at a dose of 375 mg/ ^m2 for a total of eight doses or once, followed by seven additional doses of 500 mg/ ^m2 . The therapy resulted in an ORR of 31% (9% CR, 22% PR), with no difference in efficacy between the two treatment regimens. The ORR was 37% in patients with diffuse large cell lymphoma (N = 30) and 33% in patients with mantle cell lymphoma (N = 12).

Combined use of Rituximab® treatment and CHOP chemotherapy

In a further study, 31 patients with intermediate- or high-grade NHL (19 women, 12 men, median age 49) received Rituximab® on the first day of each of six 21-day CHOP cycles [Link et al.: Proceedings of the American Society of Clinical Oncology 17, 3a (1998)]. Of the 30 evaluable patients, 19 achieved CR.

- 45 (63%), 10 had a PR (33%) (ORR = 96%). The treatment was well tolerated by patients and may be more effective than Rituximab® or CHOP therapy alone.

In collaboration with the Cancer Treatment and Diagnosis Group of NCI and IDEC Pharmaceuticals Corporation, we are conducting research to explore additional indications for Rituximab® therapy. We are conducting studies comparing CHOP and CHOP + Rituximab® therapy in older (>60 years) patients with mixed cell, diffuse large cell, and immunoblastic large cell NHL (planned N.=630). The trial will also include a second randomization to Rituximab® maintenance therapy and no maintenance therapy.

A phase III trial of Rituximab® + CHOP in 40 previously untreated patients with mantle cell lymphoma is also underway at Dane Farber. Rituximab® is administered on the first day of six 21-day treatment cycles, and CHOP is administered on the first and third days. The evaluation of this trial has been completed. A phase II trial of CHOP followed by Rituximab® in newly diagnosed follicular lymphoma patients is also underway at SWOG. The results of these latter two trials are currently being evaluated.

The AIDS Malignancy Consortium is currently conducting a trial comparing the effects of Rituximab® + CHOP and CHOP alone.

- 46 evaluators are planning to conduct a phase II trial in 120 patients with HIV-related NHL.

Use of rí tvxi mah® in cases of tumor recurrence following myeloablative therapy

Rituximab® treatment has shown promising preliminary results in patients with relapsed intermediate-grade NHL after high-dose autologous PBSC support. Six of the seven patients responded to therapy (1 CR and 5 PR), one had stable disease, and all were well tolerated [Tsai et al.: Blood 92, 415a #1713 (1998)].

Safety experience

A database of adverse events and clinical laboratory values from 315 patients in five US single-agent studies was created to describe the safety profile of Rituximab® in patients with low-grade or follicular NHL. The vast majority of adverse events were infusion-related and decreased with repeated infusions. The most common adverse events were fever (49%), chills (32%), nausea (18%), syncope (16%), headache (14%), edema (13%), pruritus (10%), and occasionally hypotension (10%) and bronchospasm (8%). During treatment (within 30 days of the last dose), 10% of patients experienced grade 3 or 4 adverse events, primarily infusion-related or hematological. Thrombocytopaenia (< 50,000 platelets/mm ³ ) in

- 47 • ···?·· ·· 1.3% of patients, neutropenia (< 1000/mm ³ ) in 1.9%, and anemia (< 8 g/dL) in 1.0%. Although Rituximab® caused a decrease in B-cell counts in 70-80% of patients, an abnormal decrease in serum immunoglobulins was observed in only a few of them and the frequency of infections was not increased.

Hypotension requiring interruption of Rituximab® infusion occurred in 10% of patients, with 1% experiencing grade 3 or 4. Aqueous infiltrates were reported in 13% of patients, with one being considered severe. Bronchospasm occurred in 8% of patients, and 2% were treated with bronchodilators. Bronchiolitis obliterans was reported in one case. The majority of patients did not experience any further infusion-related toxicities with the second or subsequent infusions. The proportion of patients experiencing adverse reactions during retreatment was similar to that experienced with the first treatment [Davis et al.: Blood 92 (10 suppl. 1) 414a (1998)].

Four patients experienced arrhythmias during Rituximab® infusion. One patient had to discontinue treatment due to ventricular and supraventricular tachycardia. The other three patients experienced trigeminal neuralgia (N = 1) and irregular pulse (N = 2), and did not require discontinuation of treatment. One patient with a previous myocardial infarction developed angina during the infusion and developed a myocardial infarction four days later.

- 48 • ······ · · ····*· · ·*

The incidence of adverse events and grade 3 or 4 adverse events was higher in patients with bulky disease. Dizziness, neutropenia, thrombocytopenia, myalgia, anemia, and chest pain were more common in patients with tumors larger than 10 cm. Grade 3 or 4 neutropenia, anemia, hypotension, and dyspnea were more common in patients with bulky disease than in those with tumors smaller than 10 cm [Davis et al.: Blood 92 (10 suppl. 1) 414a (1998)].

Approximately 17,000 patients have been treated since the FDA approved Rituximab® for the treatment of relapsed or refractory, low-grade, or follicular NHL in 1997. In May 1998, eight postmarketing reports of fatal adverse events associated with Rituximab® were summarized. Of the eight deaths, seven occurred during the first infusion. In two of the eight cases, the cause of death was unknown or not reported. Six of the deaths were due to severe respiratory failure with hypoxia, pulmonary edema, or adult respiratory distress syndrome (ARDS). One patient had a pretreatment lymphocyte count of 600,000/mm ³ , another had a creatinine level of 8, a third had a respiratory rate of 40/min, and a fourth had a

- 49 • ···}·· · pancytopenia was observed. Patients with extensive malignancy or with a high number of circulating malignancies may be at increased risk of adverse reactions and should be monitored closely with each infusion.

Most of the adverse reactions described here were already known from previous clinical trials with Rituximab®. An exception was an infusion-related syndrome caused by rapid lysis of tumor tissue, which was observed in six patients with a large number of circulating tumor cells [Byrd et al.: Blood 92 (10 suppl. 1), 106a (1998); Jensen et al.: Annals of Hematology 77, 89 (1998)]. This syndrome was characterized by fever, chills, bronchospasm with hypoxemia, a rapid decrease in peripheral lymphocyte counts, laboratory values consistent with tumor tissue destruction, and transient, severe thrombocytopenia. Two of the patients he treated had B-prolymphocytic leukemia, two had chronic lymphocytic leukemia, and one had mantle cell lymphoma or transformed NHL, all of whom had elevated circulating lymphocytes, large lymph nodes, and organ enlargement. Although five of the six patients required hospitalization, their symptoms resolved and subsequent Rituximab® treatments were well tolerated. One patient refused further treatment and died two weeks later due to worsening of his disease.

- 50 • · ···· ♦ · ·«···«« · ·♦

Another publication reported that seven CLL and one mantle cell lymphoma patients with lymphocyte counts greater than 10 x 10 ⁹ /L experienced tumor lysis syndrome after the first Rituximab® infusion [Winkler et al.: Blood 92, 285b #4228 (1998)].

Radioimmunotherapy using a combination of 90yttrium-labeled anti-CD20 antibody and Rituximab

Another option for the treatment of non-Hodgkin lymphoma is the combination of a radiolabeled anti-CD20 antibody (IDEC-Y2B8) and Rituximab®. IDEC-Y2B8 ( ⁹⁰ Y-ibritumomab tiuxetan) is a murine IgGi kappa anti-CD20 antibody covalently linked to ⁹⁰ Y via the chelating agent MX-DTPA. Rituximab® (250 mg/m ² ) is administered prior to the administration of IDECY2B8 to reduce the number of peripheral B lymphocytes and improve the biodistribution of the radiolabeled antibody.

In a recently published phase I/II study, patients with low- (N = 34) or intermediate-grade NHL (N = 14) or mantle cell lymphoma (N = 3) were treated with IDECY2B8 [Witzig et al.: Journal of Clinical Oncology, submitted for publication (1999); Wiseman et al.: Blood 92, 417a (1998); Witzig et al.: Blood 92, 417a (1998)]. The median age of the patients treated was 60 years, 71% were male, and 96% were white. Of the 51 patients with relapsed or refractory NHL, 34 (67%)

- 51 responded to a single dose of IDEC-Y2B8 at 0.2, 0.3, or 0.4 mCi/kg. The ORR was 82% (24/32) in patients with low-grade or follicular NHL and 43% (6/14) in patients with intermediate-grade lymphoma. Patients with mantle cell lymphoma did not respond to treatment.

A randomized phase III trial comparing IDEC-Y2B8 with Rituximab® (375 mg/ ^m2 once weekly for four doses) in patients with low-grade follicular or transformed NHL is currently underway. A phase III trial in patients with relapsed NHL resistant to Rituximab® is also ongoing.

Summary

In the absence of a curative therapy for non-Hodgkin lymphoma, the goal of treatment is to suppress the disease for an appropriate period of time and to alleviate the associated symptoms without excessive toxic side effects. Rituximab® treatment is a short-term, 21-day outpatient procedure that has limited adverse side effects in the majority of patients. In clinical trials, 50% of patients with relapsed or chemotherapy-resistant, low-grade or follicular NHL with evaluable data achieved complete or partial remission. This remission was maintained in the absence of maintenance therapy, with preliminary results showing a median TTP of 13.2 months and a median DR of 11.6 months for responders.

- 52 « ··♦ · · ·♦* .:.. .·.· *·γ

Rituximab® is a safe and effective drug approved for the treatment of patients with relapsed low-grade or follicular B-cell NHL. It has significant clinical activity, a novel mechanism of action, and provides an advantage in response rates and duration of improvement over other available treatments. Ongoing studies will demonstrate the feasibility of alternative Rituximab® regimens and the applicability of Rituximab® in the treatment of other CD20+ B-lymphocyte-derived malignancies.

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Claims (31)

PATENT CLAIMS 1. Use of an anti-CD20 antibody for the preparation of a composition for the treatment of a patient with relapsed B-cell lymphoma. 2. Use according to claim 1 for the preparation of a composition for treating a patient previously treated with an anti-CD20 antibody. 3. Use according to claim 1 for the preparation of a composition for the treatment of a patient who has previously undergone a bone marrow or stem cell transplant. 4. Use according to claim 1 for the preparation of a composition for the treatment of a patient who has previously undergone radiotherapy. 5. Use according to claim 1 for the preparation of a composition for the treatment of a patient who has previously undergone chemotherapy. 6. The use according to claim 5, for the preparation of a composition for treating a patient who has undergone any of the following chemotherapy: CHOP, ICE, mitosantrone, cytarabine, DVP, ATRA, idarubicin, Hoelzer chemotherapy regimen, La La chemotherapy regimen, ABVD, CEOP, 2-CdA regimen, FLAG & IDA, VAD, Μ & P, weekly C, ABCM, MOPP or DHAP regimen with or without concomitant G-CSF. 7. Use of an anti-CD20 antibody in the preparation of a composition for the treatment of a patient with B-cell lymphoma - 63 patients in whom previous use of a targeted anti-CD20 antibody did not result in detectable tumor remission or relapse. 8. The use of claim 7 for the preparation of a composition for treatment for a period of about 1 week to about two years following administration of the chimeric anti-CD20 antibody. 9. The use of claim 8 for the preparation of a composition for treatment for a period of about 1 week to about nine months following administration of the chimeric anti-CD20 antibody. 10. The use of claim 1, wherein the anti-CD20 antibody is a chimeric anti-CD20 antibody. 11. The use according to claim 10, wherein the chimeric anti-CD20 antibody is C2B8 (Rituximab®). 12. Use of at least one anti-CD20 antibody and at least one cytokine for the preparation of a composition or compositions for the synergistic treatment of B-cell lymphoma. 13. The use of claim 12, wherein the cytokine is any one of the following: α-interferon, γ-interferon, IL-2, GM-CSF, or C-CSF. 14. The use of claim 12, wherein a first composition comprising a cytokine and a second composition comprising an anti-CD20 antibody are used, or a composition comprising an anti-CD20 antibody and a cytokine together. 15. The use of claim 12, wherein the anti-CD20 antibody is a chimeric antibody. 16. The use according to claim 15, wherein the chimeric anti-CD20 antibody is C2B8 (Rituximab®). 17. Use of an anti-CD20 antibody for the manufacture of a composition for the treatment of B-cell lymphoma prior to, concurrently with, or subsequent to the administration of a chemotherapy regimen. 18. The use of claim 17, wherein the chemotherapy regimen is any of the following: CHOP, ICE, mitosantrone, cytarabine, DVP, ATRA, idarubicin, Hoelzer chemotherapy regimen, La La chemotherapy regimen, ABVD, CEOP, 2-CdA, FLAG & IDA, VAD, M & P, weekly C, ABCM, MOPP or DHAP with or without concomitant G-CSF. 19. The use of claim 17, wherein the anti-CD20 antibody is a chimeric antibody. 20. The use of claim 19, wherein the chimeric anti-CD20 antibody is C2B8 (Rituximab®). 21. Use of an anti-CD20 antibody for the preparation of a composition for the treatment of B-cell lymphoma prior to, concurrently with, or following bone marrow or stem cell transplantation. 22. The use of claim 21, wherein the anti-CD20 antibody is a chimeric antibody. 23. The use according to claim 22, wherein the chimeric anti-CD20 antibody is C2B8 (Rituximab®). 24. Use of an anti-CD20 antibody for the preparation of a composition for reducing the number of CD20+ tumor cells in the bone marrow or among stem cells before or after myeloablative therapy. 25. The use of claim 24, wherein the anti-CD20 antibody is a chimeric antibody. 26. The use of claim 25, wherein the chimeric anti-CD20 antibody is C2B8 (Rituximab®). 27. The use of claim 1, wherein the B-cell lymphoma is any of the following: low-grade/follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade/follicular NHL, intermediate-grade diffuse NHL, chronic lymphocytic leukemia (CLL), high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleft NHL, NHL with extensive tumor tissue formation, mantle cell lymphoma, AIDS-associated lymphoma, or Waldenstrom's macroglobulinemia. 28. The use of claim 12, wherein the B-cell lymphoma is any of the following: low-grade/follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade/follicular NHL, intermediate-grade diffuse NHL, chronic lymphocytic leukemia (CLL), high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleft NHL, NHL with extensive tumor tissue formation, mantle cell lymphoma, AIDS-associated lymphoma, or Waldenstrom's macroglobulinemia. 29. The use of claim 17, wherein the B-cell lymphoma is any of the following: low-grade/follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade/follicular NHL, intermediate-grade diffuse NHL, chronic lymphocytic leukemia (CLL), high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleft NHL, NHL with extensive tumor tissue formation, mantle cell lymphoma, AIDS-associated lymphoma, or Waldenstrom's macroglobulinemia. 30. The use of claim 21, wherein the B-cell lymphoma is any of the following: low-grade/follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade/follicular NHL, intermediate-grade diffuse NHL, chronic lymphocytic leukemia (CLL), high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleft NHL, NHL with extensive tumor tissue formation, mantle cell lymphoma, AIDS-associated lymphoma, or Waldenstrom's macroglobulinemia. 31. The use of claim 24, wherein the B-cell lymphoma is any of the following: low-grade/follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade/follicular NHL, intermediate-grade diffuse NHL, chronic lymphocytic leukemia (CLL), high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleft NHL, bulky tumor tissue - 67 NHL with formation, mantle cell lymphoma, AIDS-associated lymphoma or Waldenstrom's macroglobulinemia. The authorized person: DANUBE Patent and Trademark Office Ltd. Dr. Árpád Pethő^ Patent Attorney